Diabetes mellitus is the most prevalent metabolic disease, and -cell apoptosis contributes to decreases in -cell mass and function during the evolution of diabetes. However, the mechanism(s) that contribute to -cell apoptosis are not well-understood. Our hypothesis in the 1st grant period was that the Group VIA Ca2+- independent phospholipase A2 (iPLA2) participates in ER stress-induced -cell apoptosis. We find that (a) ER stress induces iPLA2 activation, ceramide generation via neutral sphingomyelinase (NSMase), and -cell apoptosis, (b) these outcomes are suppressed by inhibition of iPLA2 or NSmase, (c) iPLA2 -null islets are less and iPLA2 -tansgenic (Tg) islets more sensitive to ER stress-induced apoptosis; that ER stress (d) increases iPLA2 protein/activity in the ER and mitochondria, and (e) activates the mitochondrial apoptotic pathway via the iPLA2 -ceramide axis; and (f) ER stress-prone Akita -cells and islets from pre-diabetic NOD mice express higher levels of iPLA2 than WT cells and islets, (g) STZ-induced hyperglycemia is accelerated in iPLA2 -Tg mice, and (h) cytokines induce ceramide generation, loss in , and apoptosis in islet-cells that are all suppressed by iPLA2 inactivation. During the 2nd grant period, we propose to examine the mechanism of involvement of iPLA2 and iPLA2 -derived lipid mediators in -cell apoptosis under the following Aims: Aim 1 will examine iPLA2 activation and lipid changes in -cells undergoing apoptosis. Hyperglycemia and cytokines promote -cell apoptosis, in part, by inducing ER stress and the dependence of this process on iPLA2 will be assessed. Aim 2 will examine the mechanism of iPLA2 and ceramide-generating pathway induction. The roles of lipid mediators, SREBPs, ROS, and GSH on iPLA2 and NSMase expression and the affects of mutating the lipase sequence in iPLA2 on ceramide generation will be examined. Aim 3 will examine the role of iPLA2 in ER-mitochondria crosstalk. The affects of genetic modulation of iPLA2 expression, ROS and GSH levels, NSMase expression, mutations in iPLA2 , and organelle-specific iPLA2 expression on activation of the mitochondrial apoptotic pathway will be examined. Aim 4 will examine if in vivo modulation of iPLA2 expression alters islet -cell sensitivity to ER stress. iPLA2 -null mice will be crossed with Akita mice and iPLA2 -Tg mice with CHOP-null mice and the development of ER stress monitored. Aim 5 will determine if iPLA2 contributes to -cell apoptosis during the evolution of autoimmune DM. The dependence of cytokine-induced -cell apoptosis on iPLA2 and nitric oxide pathway and of STZ-induced -cell apoptosis and hyperglycemia on iPLA2 expression will be determined. Apoptosis, flow cytometry, immunoblotting, real time-PCR, enzymatic activity, confocal microscopy, molecular biology, and mass spectrometry protocols will be utilized in these studies.